AHNAK-modified microbubbles for the intracranial delivery of triptolide: In-vitro and in-vivo investigations.

Autophagic dysfunction related cascade events might induce the accumulation of α-synuclein (αSyn) in Parkinson's disease (PD). Triptolide (T₁₀) has been reported as a potential autophagy inducer but limited by hepatotoxicity, low solubility and rapid metabolism. In this study, a novel AHNAK-targeted microbubbles integrated with T₁₀ (T₁₀-AHNAK-MBs) was developed to alleviate motor deficit in rAAV2/5-wild type and A53T mutant αSyn transfected PD mouse model. AHNAK facilitated the accumulation of microbubbles (MBs) near the cerebral vessel wall. Furthermore, bubble cavitation caused by focused-ultrasound (FUS) exposure could simultaneously induce drug release and blood-brain-barrier opening in the area of interest. The results of western blotting, thioflavin S staining, immunofluorescence, ELISA and behavior test demonstrated that T₁₀-AHNAK-MBs with FUS exposure (T₁₀-AHNAK-MBs-FUS) could significantly delivery more T₁₀ into substantia nigra, promote clearance of various forms of αSyn, reduce tyrosine hydroxylase positive neuron loss, restore dopamine secretion, and eventually alleviate motor deficits, along with largely reduced adverse effects. The analyses of autophagic markers suggested that autophagy lysosome pathway (ALP) might dominate the T₁₀-induced αSyn degeneration, including the oligomers and pre-formed fibrils. Thus, T₁₀-AHNAK-MBs-FUS constitutes a promising strategy against the motor deficits in PD by promoting clearance of pathogenic αSyn aggregates via inducing ALP.